Journal: The Journal of Immunology Author Choice

Article Title: Macrophage-intrinsic and IL-9–dependent arginine metabolism promotes lung tumor growth

doi: 10.1093/jimmun/vkag026

Figure Lengend Snippet: IL-9–dependent regulation of ARG1 dysregulates lung polyamine concentration in tumor-burdened lungs. (A–D) B16 melanoma cells were intravenously injected into Il9r fl/fl Lyz2-Cre and control mice ( n = 3 to 5). (A) Analysis of tumor burden 21 days after injection. (B–D) Identification and analysis of lung macrophages by flow cytometry. (E–H) Il9r fl/fl Lyz2-Cre and control mice were intravenously injected with LLC cells. (E) Analysis of tumor burden 21 days after injection ( n = 3 to 5). Identification and analysis of lung macrophage populations ( n = 3 to 5). (F–H) Flow cytometric analysis of lung macrophages. (I, J) Quantification of arginine-derived metabolites from human lung and tumor tissue and serum ( n = 5 to 6). (K–N) Arginine metabolite quantification from tumor-bearing (i.v. B16 or LLC) WT, Il9r −/− , and Il9r fl/fl Lyz2-Cre mice. (K, L) Polyamine quantification of lung lysate ( n = 4) and BALF ( n = 4) from tumor-bearing WT and Il9r −/− mice. (M, N) Polyamine quantification of lung lysate ( n = 3 to 4) and BALF ( n = 4) from Il9r fl/fl Lyz2-Cre mice from 2 independent experiments. Data represent the mean ± SEM. Statistical analyses were performed using 2-tailed Mann–Whitney test (A–H), paired Student t-tests (I), and Welch t-test (J–N). Data are representative of 2 independent experiments that used 3 to 5 mice per group per experiment.

Article Snippet: B16-F10 melanoma (CRL-6475) and Lewis lung carcinoma (LLC) (LL/2, CRL-1642) cell lines were purchased from American Type Culture Collection (ATCC).

Techniques: Concentration Assay, Injection, Control, Flow Cytometry, Derivative Assay, MANN-WHITNEY

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Identification of 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK)-binding small-molecule compounds by high-throughput screening (HTS). (A) Small molecule compounds are printed onto homemade phenyl-isocyanate-functionalized glass slides and immobilized through amino, hydroxyl, and thiol groups as well as other interactions. In oblique-incidence reflectivity difference (OI-RD) scanning of small-molecule microarrays (SMMs), successful binding of the target protein at a specific location with small-molecule compounds increases the molecular layer thickness, which OI-RD detects as white spots. The binding of proteins to small molecule compounds is usually coupled mediated by nucleophilic groups (hydroxyl, amino, and thiol). Interactions such as hydrogen bonding will be further formed thereby mediating protein interactions with small molecule compounds. No spot is observed if binding does not occur. Created with www.BioRender.com . (B) Categories of the compound library. Approximately 4389 compounds, including 1678 Food and Drug Administration (FDA)-approved drugs, 1456 natural products, and 1255 known inhibitors, were printed on the chip. (C) A representative image showing the binding of the recombinant AMPK protein to compounds printed on an HTS chip. (D) A real-time OI-RD assay depicting the association‒dissociation curves of surface-immobilized Ribavirin binding to the recombinant AMPK protein. (E) A surface plasmon resonance (SPR) experiment was used to evaluate the interaction kinetics and binding affinity between Ribavirin and AMPK. (F, G) Quantitative real-time polymerase chain reaction (qRT-PCR) measurement of Ampk transcripts in mouse Lewis lung carcinoma (LLC) cells (F) or human non-small cell lung cancer cells (A549) (G). The data are presented as the relative fold induction of Ampk/actin transcripts. Arrows indicate groups with significantly reduced Ampk transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Binding Assay, High Throughput Screening Assay, Drug discovery, Recombinant, SPR Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: The impact of Ribavirin on inflammatory factors is regulated downstream of the 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway. (A, B) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of interleukin-6 ( Il - 6 ) transcript levels in mouse Lewis lung carcinoma (LLC) cells (A) and human non-small cell lung cancer cells (A549) (B) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Il - 6/actin transcripts. Arrows indicate groups with significantly reduced Il - 6 transcripts. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001. (C, D) qRT-PCR analysis of Tnf α transcript levels in LLC (C) and A549 (D) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Tnf α/actin transcripts. Arrows indicate groups with significantly reduced Tnf α transcripts. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (E, F) qRT-PCR analysis of C-X-C motif chemokine ligand 10 ( Cxcl10 ) transcript levels in LLC (E) and A549 (F) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Cxcl10/actin transcripts. The arrows indicate groups with significantly reduced Cxcl10 transcript levels. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (G, H) qRT-PCR analysis of C–C motif chemokine ligand 5 ( Ccl5 ) transcript levels in LLC (G) and A549 (H) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Ccl5/actin transcripts. Arrows indicate groups with significantly reduced Ccl5 transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗ P < 0.05. mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Ribavirin suppresses lung cancer cell growth both in vitro and in vivo . (A, B) Viability of mouse Lewis lung carcinoma (LLC) cells (A) and human non-small cell lung cancer cells (A549) (B) cells treated with various concentrations of Ribavi rin. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. (C, D) Viability of LLC (C) and A549 (D) cells following treatment with 10 μM Ribavirin or the control for 1–5 days. The data shown are the means ± SEM from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (E, F) Colony formation of LLC (E) and A549 (F) cells after treatment with 10 μM Ribavirin or the control. The data shown are the means ± SEM from n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (G) Tumor anatomy of LLC subcutaneous tumor-bearing mice. (H) Tumor volume of LLC subcutaneous tumor-bearing mice. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 5 mice per group, with differences denoted by ∗ P < 0.05. (I) Tumor weights of LLC subcutaneous tumor-bearing mice. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 8 mice per group, with differences denoted by ∗∗ P < 0.01. PBS: phosphate buffered saline.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: In Vitro, In Vivo, Control, Saline

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: The interaction of Ribavirin with 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK). (A) Biotin was conjugated to the amide residue of Ribavirin to produce biotin-conjugated Ribavirin. (B) Immunoblotting (IB) of the streptavidin precipitates of the mixture of biotin or biotin-Ribavirin and the lysates of human embryonic kidney epithelial cells (HEK293T) cells transfected with Flag-AMPK. The data shown are representative of n = 3 biological replicates. (C–F) The molecular docking assay illustrates the interaction between Ribavirin and AMPK, identifying the key amino acids involved (C); the blue protein represents AMPK (D), the green small molecule represents Ribavirin (E), and the yellow dashed lines represent the hydrogen bonds formed (F). A binding energy below −5 suggests high reliability of the docking model. (G, H) Immunoblotting of phosphorylated AMPK (p-AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lysates of mouse Lewis lung carcinoma (LLC) cells (G) and human lung adenocarcinoma cells (PC9) (H) cells in the presence of Ribavirin (10 μg/mL). The data shown are representative of n = 3 biological replicates. (I, J) Immunoblotting of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (p-4EBP1) and GAPDH in the lysates of LLC (I) and PC9 (J) cells in the presence of Ribavirin (10 μg/mL). The data shown are representative of n = 3 biological replicates. (K, L) Quantitative real-time polymerase chain reaction (qRT-PCR) measurement of 4ebp1 (K) and Mtor (L) transcripts in LLC cells in the presence of phosphate buffered saline (PBS) or the indicated concentrations of Ribavirin (10 μm). The data are presented as the fold induction of 4ebp1/Gapdh transcripts and Mtor/Gapdh transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. ns: not significant; IP: immunoprecipitation; mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Residue, Western Blot, Transfection, Docking Assay, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Saline, Immunoprecipitation

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Ribavirin has synergistic effects on 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) downregulation. (A) Three AMPK-targeting short hairpin RNAs (shRNAs) were screened to evaluate their knockdown efficiency and suppress AMPK expression in lung cancer cells. The data shown are representative of n = 3 biological replicates. (B) Tumor anatomy of mouse Lewis lung carcinoma (LLC) cells subcutaneous tumor-bearing mice in different treatment groups. (C) Tumor volume of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± standard error of the mean (SEM); n = 8 mice per group, with differences denoted by ∗ P < 0.05 and ∗∗ P < 0.01. (D) Tumor weights of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 8 mice per group, with differences denoted by ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (E) Schematic illustration of the mechanism by which Ribavirin regulates AMPK activation. AMPK is phosphorylated in response to an increased AMP/adenosine triphosphate (ATP) or adenosine diphosphate (ADP)/ATP ratio, influencing cell growth and proliferation by inactivating mechanistic target of rapamycin complex 1 (mTORC1). Ribavirin exerts its regulatory effect by inhibiting AMPK phosphorylation. Created with www.BioRender.com . shControl: non-targeting control short hairpin RNA; shAMPKα: AMPKα-targeting short hairpin RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PBS: phosphate buffered saline; mTOR: mechanistic target of rapamycin; Raptor: regulatory-associated protein of mTOR; Deptor: DEP domain-containing mTOR-interacting protein; PRAS40: proline-rich Akt substrate of 40 kDa; mLST8: mammalian lethal with SEC13 protein 8; 4EBP1:eukaryotic translation initiation factor 4E-binding protein 1; P: phosphorylation.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Knockdown, Expressing, Activation Assay, Phospho-proteomics, Control, shRNA, Saline, Binding Assay